Alternative pre-messenger RNA splicing is a critical means of eukaryotic gene regulation that allows a single gene to produce a variety of mRNAs and proteins. Many proteins important for neuronal development and activity are functionally diversified through the differential inclusion of alternative exons. In spite of its importance to neuronal function and disease, the mechanisms controlling alternative splicing are poorly understood. We propose to continue our studies of neuronal exon splicing with a focus on four regulatory proteins. Polypyrimidine Tract Binding Protein (PTB) and its neuronal homolog nPTB are splicing repressers for multiple exons. The Fox-1 and Fox-2 proteins are enhancers of a different but overlapping set of exons. Previously, the regulation of the neuron-specific N1 exon of c-src was reconstructed in vitro. We will use this system to analyze how PTB represses spliceosome assembly, and identify its target interactions. Neuronal PTB does not repress the splicing of N1 and other neuronal exons. Experiments will examine how this highly homologous protein differs in activity. The Fox proteins activate N1 and other neuronal exons through an important splicing enhancer element UGCAUG. The mechanism of this splicing stimulation will be examined using both in vitro and in vivo approaches. The biology of these regulators in differentiating neurons will be explored through their depletion or mis-expression in cells. Groups of exons controlled by these factors will be identified in microarray experiments and examined for common features and function in common regulatory pathways. The complex posttranscriptional regulation of nPTB by PTB in neurons and glia will also be a focus. Through these experiments, we hope to understand both the mechanisms of these proteins' action, and the role they play in neuronal cell biology. The understanding of alternative splicing is essential to our understanding of multiple forms of genetic disease. Spinal Muscular Atrophy, Myotonic Dystrophy, and Prefrontal Dementia are neurologic disorders of splicing regulation. Many human disease mutations alter splicing regulatory elements to produce aberrant proteins. For these diseases to be approached therapeutically, much more information is needed on the mechanisms of splicing regulation and its role in neuronal function.